Lens and associatable flow cell

ABSTRACT

Improvements in a biosensor are disclosed. A biosensor includes a waveguide, at least a portion of which is substantially planar. One or more reservoirs may be formed adjacent to a chemistry-bearing surface of the waveguide. The biosensor may include a gasket to form a seal between the waveguide and side walls of the reservoir. A sample solution may be introduced into the reservoir or otherwise onto the surface of a waveguide through an input port. Waveguides of varying composition (e.g., plastic, quartz, glass, or other suitable waveguide materials) may be used in the biosensor. Also disclosed is a sled-shaped waveguide, which includes a planar portion and a lens at an end thereof and angled relative thereto for coupling light into the waveguide.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a continuation of application Ser. No.09/608,714, filed Jun. 30, 2000, now U.S. Pat. No. 6,356,676, issuedMar. 12, 2002, which is a divisional of application Ser. No. 09/142,948,filed Sep. 18, 1998, now U.S. Pat. No. 6,108,463, issued Aug. 22, 2000,which is an application under 35 U.S.C. § 371 of PCT/US 97/04398 filedon Mar. 19, 1997 claiming priority from U.S. Provisional PatentApplication No. 60/022,434 filed on Aug. 8, 1996 and U.S. ProvisionalPatent Application No. 60/013,695 filed on Mar. 19, 1996.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to components of a diagnosticapparatus, and more particularly to an improved biosensor having a lens(“waveguide”) and associated flow cell.

[0004] 2. State of the Art

[0005] International Application No. PCT/US94/05567 (InternationalPublication No. 94/27137, published Nov. 24, 1994) to the University ofUtah Research Foundation discloses an apparatus for multi-analytehomogeneous fluoroimmunoassays. In one embodiment, the applicationdiscloses an apparatus which uses a biosensor having a planar waveguidesandwiched, with an associated gasket, between two plates (FIGS. 3A-3Cof Internat'l Publ. No. 94/27137). The inner edges of the gaskets serveas walls for a reaction reservoir or well. Fluorescence-emitting tracermolecules are bound to the waveguide surface and are excited by anevanescent field penetrating into the adjacent solution from a lightbeam propagated within the waveguide, the beam being introduced at, forexample, a front end of the waveguide. In the reaction reservoir, aliquid (e.g., serum or blood) is introduced and is allowed to admix withcapture molecules associated with the waveguide surface (e.g., by“coating chemistry” as disclosed on pages 32 to 33 of the internationalapplication). The emitted fluorescence is then directly collected fromthe zone of evanescent penetration. In one particular embodiment, thebiosensor has transparent walls which define the reservoirs. (E.g.,FIGS. 11A-C of Internatl. Publn. No. 94/27137). The application alsodiscloses integrally formed or molded biosensors. (E.g., FIGS. 12A, 12B& 13 of Internatl. Publn. No. 94/27137).

[0006] Unfortunately, the waveguide portion of integrally formed ormolded biosensors may exhibit deformation upon fabrication, or warpingduring storage or temperature changes. Also, gaskets may not reliablyseal or are not always sufficiently inert to reactants, and thus mayinterfere with the desired analysis.

[0007] It would be desirable to have a biosensor having reservoirs withinert walls, the walls being readily detachable from the waveguide sothat one waveguide could be readily exchanged for another.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention includes a biosensor with a reservoir orreservoirs, the biosensor including a waveguide placed (e.g.,“sandwiched”) between a plurality of members such as plates, at leastone of the members being formed to define the walls of the reservoir orreservoirs where the reaction to be analyzed takes place. The reservoirwalls are preferably an inert, opaque material such as a passivatedmetal (e.g., black anodized aluminum). Although the biosensor mayinclude a gasket, the gasket is associated with the plurality of membersand waveguide in such a way (e.g., by recessing the gasket into achannel formed into a metal plate) so that the gasket does not form anysignificant portion of the reservoir wall. Waveguides of varyingcomposition (e.g., plastic, quartz, glass or siliconoxynitride) may beassociated with the members to form the biosensor. A lens or lenses maybe integrated with the waveguide. The metal plate of the biosensor hasinput and output ports for infusing, draining, or oscillating the liquidto be analyzed in the reaction reservoir.

[0009] Due to the sandwiching of the waveguide in between the members,the planar waveguide is generally less distorted than that of anintegrally formed biosensor. A reaction to be analyzed is not interferedwith due to the use of opaque, inert metal to structurally define thereservoir.

[0010] The biosensor design is advantageously configured to interactwith a flat waveguide having a rear integrated lens design for readinglight passing through the waveguide (not fluorescent/evanescent light,but reading the core laser beam light) to monitor coupling efficiencyand beam quality. The invention thus also includes a flat waveguideassociated with a rear lens to couple light out of the waveguide (and abiosensor using such a lens) to serve as a quality control measure, thusinsuring that the biosensor is properly placed and that the light sourceis working.

[0011] The invention also includes orienting the biosensor in aparticular position relative to an optical reading device and laserwhich increases the performance of the biosensor to the point where,surprisingly, whole blood can be quickly analyzed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0012] In the drawings, which depict presently preferred embodiments ofthe invention and in which like reference numerals refer to like partsin different views:

[0013]FIG. 1 depicts an enlarged bottom view of the flow cell top whichmay be used with the invention.

[0014]FIG. 2 depicts an enlarged section view of the flow cell top ofthe preceding figure along section line 2-2.

[0015]FIG. 3 depicts an enlarged section view of the flow cell top ofFIG. 1 along section line 3-3.

[0016]FIG. 4 depicts an enlarged, exploded, perspective view of abiosensor according to the invention.

[0017]FIG. 5 depicts an enlarged, side view of a laminated gasket whichinteracts with the flow cell top of FIG. 1.

[0018]FIG. 6 depicts an enlarged view of a plastic, molded flatwaveguide with integrated input and output coupling lenses.

[0019]FIG. 7 is an enlarged side view of the waveguide of the precedingfigure.

[0020]FIG. 8 is a schematic diagram of a fluorescent assay apparatususeful for practicing the invention, showing the flow cell assembly ofFIG. 4 in a particularly useful orientation with respect to the earth.

[0021]FIG. 9 is a stylized, enlarged side view of a portion of awaveguide and biochemical components of a competition immunofluorescentassay according to the invention.

[0022]FIG. 10 depicts an enlarged, exploded, perspective view of theflow cell portion of a biosensor according to the invention.

[0023]FIG. 11 depicts an enlarged perspective view of a gasket for usewith the invention.

[0024]FIG. 12 depicts an enlarged top view of a second frame member(“flow cell bottom”) for association with the flow cell top of FIGS. 1through 3.

[0025]FIG. 13 depicts an enlarged side view of the second frame memberof the preceding figure.

[0026]FIG. 14 depicts an enlarged perspective view of the registrationplate forming part of the flow cell assembly of FIG. 4.

[0027]FIG. 15 depicts an enlarged top view of the stage member of theflow cell assembly of FIG. 4.

[0028]FIG. 16 depicts an enlarged side view of the stage member of thepreceding figure.

[0029]FIG. 17 depicts an enlarged view of the preceding two figures.

[0030]FIG. 18 depicts an enlarged, exploded, perspective view of abiosensor according to the invention.

[0031]FIG. 19 depicts an enlarged, stylized, side view of a portion of aplastic film waveguide having an optical diffraction grating coupler.

[0032]FIG. 20 depicts an enlarged, stylized, side view of a portion of aplastic film waveguide to which a separate optical diffraction gratinghas been associated.

[0033]FIG. 21 is a graph depicting the results of a correlationalanalysis comparing an evanescent assay (CK-MB) of the instant inventionwith a prior art device.

[0034]FIG. 22 is a graph depicting the double logarithm plot of the dataof the preceding figure showing the correlation.

[0035]FIG. 23 is a graph depicting the effect of agitation (oscillation)on a CK-MB assay on whole blood (“WB”), 50% WB, and 50% plasma using adevice according to the invention (dilution with outdated CPDAanticoagulant solution).

[0036]FIG. 24 is an enlarged, stylized illustration of wavelength- andspatially-resolved detection of fluorescence emitted from a planarwaveguide sensor using different capture molecules and tracer moleculesfor detecting different analytes of interest in a sample solution.

DETAILED DESCRIPTION OF THE INVENTION

[0037] A. Flow Cell

[0038] The flow cell top, generally 100, depicted in FIGS. 1 through 3is preferably made of a light absorbing material (e.g., a metal such asaluminum having a passivated surface such as black anodized surface).The depicted flow cell top 100 is generally plate-like, and is formed tocontain a plurality of wells or reservoirs 102, 104, 106 (for example,two to ten reservoirs).

[0039] A design with at least two individual reservoirs has significantadvantages over a single reservoir embodiment for instances when it isdesirable to measure the test sample fluorescence simultaneously withfluorescence from a “control” region on the same waveguide. For example,the level of non-specific binding to the waveguide (or non-specificfluorescence) can be subtracted from the test sample fluorescence. Also,measurement changes due to fluctuations in intensity of the excitinglight can be corrected. In a displacement assay, the “control” regioncould be a pre-loaded waveguide with no analyte present in the sample,or with a known amount of analyte. With the depicted embodiment of threeor more wells, fluorescence can be measured for both a no analytecontrol and at least one known calibration analyte sample in addition tothe “unknown” or test sample. Although, even with a single reservoir,the invention is able to analyze multiple analytes in a single sample(e.g., by use of a single waveguide in multiple experiments).

[0040] In the depicted embodiment, the reservoirs 102-106 haverespective inlet/outlet apertures 108, 110, 112, 114, 116, 118 extendingthrough the flow cell top 100 for injecting and withdrawing the liquidto be analyzed into the reservoirs 102-106. In some cases, this liquidmay be oscillated into and out of the reservoir with a pump, whichenhances the mixing of the analyte and reactant (see, e.g., FIG. 23).With oscillation, the performance (e.g., speed) of the assay isincreased. In the depicted embodiment, each port 108-118 is associatedwith its own depressed recess formed 120, 122, 124, 126, 128, 130 in theflow cell top 100.

[0041] Between the recesses associated with a particular reservoir,lateral or longitudinal channels may be formed in the flow cell top toaid in mixing the liquid contained within the reservoir (not shown).

[0042] The outer periphery of the reservoirs 102-106 are each defined byrespective walls 132, 134, 136 which are preferably integrally formedwith the rest of the flow cell top 100, although they may be a separatecomponent of the flow cell top. The inner circumferences 138, 140, 142of the walls 132-136 are made of an inert, opaque material such as aninert, opaque plastic, or a metal such as passivated, black anodizedaluminum, copper, stainless steel, or similar alloy. In the depictedembodiment, the entire flow cell top 100 is made of a metal, while inother embodiments (not shown), the flow cell may be made of anon-metallic material, and an opaque, dark material or metal sleeveplaced within the reservoirs (not shown). Material in contact with theliquid should exhibit low protein absorption properties. Accordingly, ametal, a hydrophilic non-metallic material or a hydrophobic non-metallicmaterial coated with a thin film of hydrophilic material (e.g., PEG™,PLURONICS™ or other hydrogels) may be used.

[0043] In the depicted embodiment, the apertures 108-118 associated withthe respective reservoirs 102-106 fluidically communicate the recessedportions 120-130 of the reservoirs with a pair of respective receptacles144, 146, 148, 150, 152, 154 (shown by construction lines in FIG. 1) forreceiving fluid inlet/outlet ports 156, 158 which are associated withthe flow cell top 100 (FIG. 4). Although the fluid inlet/outlet ports156, 158 will be described with regard only to one reservoir, it is tobe understood that the description applies likewise for the otherreservoirs of the flow cell (if any).

[0044] As depicted in FIG. 4, the fluid inlet/outlet ports 156, 158 maybe male threaded nipples which interact with corresponding threadedmembers (threads not shown) bored into the flow cell top 100. The openends of the nipples are in fluid communication (e.g., by tubing or otherconduit—FIG. 8) with a, for example, syringe pump (not shown). Otherfluid tight arrangements between the ports and the flow cell top can beused, so long as the sample fluid communicates to the apertures 108-118.The liquid to be analyzed (e.g., whole blood, plasma, diluents, ormixtures thereof) can thus be injected and withdrawn from the reservoirs102-106 by use of an, for example, oscillating pump (not shown).

[0045] As further depicted in FIGS. 1 and 4, the outer peripheries ofthe walls 132-136 also partially define a recess 160 formed in the flowcell top. This recess 160 is formed to accept a gasket 162 (FIGS. 4, 5 &11) which is more thoroughly described hereinafter. This gasket 162cushions placement of a waveguide onto the flow cell top 100 and impedesslippage of the waveguide when associated with the flow cell top. As isalso more thoroughly described herein, the gasket, preferably, does notserve as any part of the walls 132-136 to contain the liquid within areservoir 102-106. The flow cell may be used with a quartz waveguide or,more preferably, with the hereinafter described plastic molded waveguide164.

[0046] B. The Waveguide

[0047] As depicted in FIGS. 4, 6 & 7, a preferred waveguide 164 is aplastic molded waveguide (e.g., molded of an optical plastic) havingintegrated input 166 and output 168 coupling lenses. Such a waveguide164 is preferably sled-shaped, having a planar (optical substrate)portion 170 with first 172 and second 174 parallel plane surfaces and anedge having a thickness 176 extending therebetween (FIG. 9). At leastone of the waveguide surfaces 172 has a plurality of capture molecules240 associated therewith (e.g., immobilized thereon as depicted in FIG.9, although other methods of bringing the capture molecules intosufficiently close association with the surface may be used (e.g., byplacing a strip immunoassay onto the waveguide surface, or using afibrous “mat” with capture molecules attached to the fibers)). Thesesurfaces 172, 174 should have the best optical smoothness possible. Forexample, surface waviness and roughness spacing should lie outside arange of about 65 mn to 195 micrometers (“μm”), while spacings close to650 nm should be avoided (when 632.8 nm laser light is used). Surfaceroughness amplitude or “Ra” (theoretical surface plane to average peakor valley) should be less than 0.0065 μm (0.26 μm); however twice thisamount is still functional. The thickness will typically be between 0.20and one millimeter (“mm”), more preferably about 0.5 mm.

[0048] The edge of the planar portion has a receiving region (e.g., lens166) for receiving light to be internally propagated. In the embodimentdepicted in FIGS. 6 & 7, an input or receiving lens 166 is integrallyadapted to the waveguide adjacent the receiving region at the “front” ofthe waveguide. Other methods of optically associating the lens to theplanar portion could also be used. Surface specifications for such alens or lenses are similar to the planar or “plate” portion of thewaveguide. A maximum roughness amplitude of 0.013 to 0.025 μm (0.5 to 1μm) is preferred. Preferably, machine lines should be parallel (verticalwhen looking at lens) to the long axis of the waveguide. Surfacespecifications for the side of the part and lens ramp areas are lessstringent than the top and bottom surfaces of the plate structure.

[0049] In another embodiment (not shown), the lens (or lenses) is notintegrally associated with the waveguide, but is adapted to interactoptically with the waveguide, or multiple waveguides.

[0050] Alternatively, rather than using a lens to couple light into thewaveguide, a grating could be used. Various gratings as well as methodsfor incorporating them into a waveguide are known. See, e.g., U.S. Pat.No. 5,480,687 (Jan. 2, 1996) to Heming et al. at column 4, lines 1-10,and column 6, line 20 to column 7, line 55, U.S. Pat. No. 5,081,012(Jan. 14, 1992) to Flanagan et al., U.S. Pat. No. 5,455,178 (Oct. 3,1995) to Fattinger, U.S. Pat. No. 5,442,169 (Aug. 15, 1995) to Kunz, andU.S. Pat. No. 5,082,629 (Jan. 21, 1992) to Burgess, Jr. et al. Gratingsmay be fabricated by a number of means including but not limited to:embossing, molding, photolithography, direct etch electron beamlithography, interference lithography, and phase shift lithography.Embossed gratings are mechanically stamped or thermally imbued onto asurface and thereupon affixed to a substrate. Photolithographic gratingsare formed from the chemical development and etching of photoresist andsubstrate after masked illumination by an appropriate source.Interference and phase shift lithography are similar techniques whichallow finer resolution of etched structures than does conventionalphotolithography. Ion or particle beam methods fabricate precisegratings by directly etching or “writing” a grating substrate with astream of ions or molecular particles.

[0051] The grating itself can consist of an etched pattern of regularfeatures in a metal film coated onto the planar portion of the waveguideor the front ramp. Standard diffraction gratings such as those used inspectrometers like “replica” gratings (gratings comprised of a driedepoxy coated with metal) can be used. The use of such grating couplershelps to avoid fabrication complexities associated with the use of areceiving lens or plasma-etched gratings. The procedure for applyingsuch couplers is presently used to emboss holograms onto plastic creditcards, and, using such a process, the coupler could be mass produced ata relatively low cost.

[0052] In an alternative embodiment shown in FIG. 19, a corrugatedwaveguide with gratings 314 (>5 nanometers (“nm”) deep or thick) isassociated with (e.g., molded on, adhered to, or hot stamped onto orembossed onto) the receiving region of a plastic thin film waveguide 316(or a cast thin plastic planar waveguide) associated with (e.g., adheredto) a lower index substrate 318. Although in the depicted embodiment,the grating 314 is positioned between the thin film and the lower indexsubstrate, other orientations such as applying the grating to thesurface 172 of the thin film waveguide could also be used. Also,alternatively, the light could be directed into the waveguide from adifferent direction. In any event, the grating receives light 218 to beinternally propagated. In such a case, the waveguide portion willtypically be made of a transparent optical plastic and have a thicknessof from about 10 micrometers to about 200 micrometers, preferably about125 micrometers. In the case of extremely thin waveguide films (e.g.,about 10 to 25 μm), the resulting film may be attached to a preferablyrigid, open support structure 317 (FIG. 20). Alternately, the resultingthin film may be affixed to a supporting substrate having a lower indexof refraction than the film (FIG. 19).

[0053] From efficiency measurements, it can be determined that for anintegrated optic waveguide-fluoroimmunoassay, the most efficient etchdepths are about 1.5 times that of the grating period. For diffractionto occur in a grating, the period d should be on the order of thewavelength of light (lambda). Given the pathlength difference, δ,between the light rays from two neighboring grating features (slits,rigids, and the like), a constructive interference pattern isestablished by the light leaving the grating when δ is an integermultiple, m, of the wavelength.

δ=d(n _(t) sin Θ_(t) −n _(t) sin Θ_(l))=lambda(m)

[0054] wherein d is the grating period, Θ_(t) and Θ_(l) are thetransmitted and incident angles at the grating interface (measuredrelative to the surface normal), and n_(t) and n_(i) are the refractiveindices of the transmitting and incident mediums (i.e., the waveguideand the substrate). Using this formula, one determines that the incidentangle for coupling 632.8 nm light is 38.03° when the grating period is0.7 μm.

[0055] The angle of incidence of light from air into the lowest ordermade of the waveguide and the groove density into waveguide films can becalculated by the use of the equation above, and was determined to be4.6°, 27.4° and 57.2° for polystyrenes having densities of 2400 g/mm,1800 g/mm, and 1200 g/mm, respectively, for incident light of 632.8 μmwavelength.

[0056] In still other embodiments, laser light may be prism-coupled ontoan integrated optic waveguide (“IOW”) (not shown), end-fire coupled(i.e., direct focusing of light into the waveguide), or taper-coupled(e.g., by use of an adlayer film tapered in thickness or refractiveindex, preferably in conjunction with a grating coupler) into thewaveguide (also not shown).

[0057] In order to taper-couple light into the flow cell, a gentletapered section (e.g., either curved or linear) can be used to “funnel”light into the end of a thin planar waveguide. A well-collimated inputbeam (e.g., a laser) couples into a multi-mode waveguide (e.g., about 50μm in thickness) due to the “Law of Brightness” constraint (i.e. theproduct of the beam extent and numerical aperture is a constant throughthe taper). The taper may be also coupled with a lens.

[0058] The waveguide depicted in FIGS. 6 & 7 has a shelf or ridge 176.The ridge 176 abuts against an edge of the flow cell top when thewaveguide 164 is functionally associated with the flow cell (FIG. 4). Asshown (FIG. 1), the flow cell top 100 has two apertures 178, 180 whichinteract with registration members (“registration pins”) 182, 184 of asecond frame member (“flow cell bottom”) 186 structured to interact withthe flow cell top 100 and waveguide 164 in order to hold (“sandwich”)the waveguide in place (FIG. 4).

[0059] Laser light preferably enters the receiving lens 166 at meanangle Θ (FIG. 8). The mean angle Θ will typically vary dependent uponthe type of material used to form the waveguide and the opticalproperties of the media opposite both faces of the waveguide. When thewaveguide or waveguide layer is made of polystyrene (e.g., NOVOCOR),then the mean angle will generally be less than 32°, e.g., 15° to 25°.Typical beam widths vary from 0.5 to 2.0 mm.

[0060] On the other side of the waveguide, an outcoupling 188 interactswith the rear or output lens 168 to ensure that light is detected (FIG.8). The outcoupling 188 may be a single photodetector, multiplephotodetectors, standard CCD (charge-coupled device) or like device. Thelight passing through the waveguide 164 and received by the outcoupling188 is analyzed for quality and/or intensity. Unlike the end collectionof light described in U.S. Pat. No. 4,582,809 to Block et al. (Apr. 15,1986), in the present invention, the light may be detected at the end ofthe waveguide for two reasons. The first reason is as a quality controlmeasure. The light passing through the waveguide may be measured so thatthe operator of the device knows that the biosensor has been properlyplaced in the apparatus and that the light source is still working.Alternatively, the device may be configured so that a predeterminedstrength of light must first be detected at the rear lens 168 before theapparatus will operate, again to ensure that the flow cell assembly(“biosensor”), generally 190, has been properly placed. The secondreason for end detection involves calibration of the device to ensurethat the amount of light traveling through the waveguide is uniform and,if it is not uniform to accommodate any differences. The lightoutcoupled from the lens 168 associated with the rear of the waveguideis preferably measured over the width of the lens to ensure thatsufficient light is passing through the lens to create detectablefluorescence.

[0061] Preferably, a plastic waveguide such as that depicted in FIGS. 6& 7 will be made (e.g., injection-molded) of an optical plastic such aspolystyrene, polymethylmethacrylate (“PMMA”), polycarbonate orequivalent material, and will have a refractive index greater than 1.33(the index of water being 1.33). The size of the waveguide will dependon its desired use.

[0062] Although the front lens ramp 192 and rear lens ramp 194 are shownin a “concave” or arced position relative to one another and the planarportion 170 (FIG. 7), the ramps need not angle towards a common center,and one of the lens ramps could be angled in the opposite direction fromthe plane of the planar portion, and the ramps would fall in roughlyparallel planes (not shown).

[0063] In another embodiment (not shown), the waveguide includes alaminate of layers, one layer serving as a structural substrate, and theother (e.g., thin film SiON) serving to transmit the light, such asthose disclosed in International Application No. PCT/US96/02662(International Publication No. WO 96/26432, published Aug. 29, 1996) tothe University of Utah Research Foundation. In such an embodiment, thestructural substrate can be made of a plastic such as polystyrene, PMMA,polyvinyl chloride (“PVC”), polyimide, polyester, polyurethane,organically modified ceramics, polymers of diethylene glycol bisallylcarbonate, allyldiglycolcarbonate, polycarbonate, or equivalentmaterial. The waveguide layer is preferably an optical plastic such aspolystyrene, although it can be made of other suitable materials such asTiO₂, a mixture of TiO₂-SiO₂, SiO₂, ZnO, Nb₂O₅, Si₃N₄, Ta₂O₅, HfO₂, orZrO₂. Waveguide layers such as TiO₂, SiO₂, or Si₃N₄ can be deposited byplasma chemical vapor deposition (“PVCD”), plasma impulse chemical vapordeposition (“PICVD”) process, or the like.

[0064] C. Gasket

[0065] A gasket 162 is preferably seated between waveguide 164 and flowcell top 100 (FIGS. 4, 5 & 11). A preferred gasket 162 for use with thewaveguide 164 with integrated lenses includes a clear TEFLON™ layer 196adhered (e.g, with the use of a suitable glue or double sided tape,e.g., MACTAC No. SB 1154 available from Morgan Adhesives Co. of Stow,Ohio, USA) to a silicon rubber gasket 198 shaped to fit the recess ofthe flow cell top. Alternatively, synthetic resin polymers (i.e.TEFLON-like materials) may be used. The depicted gasket 162 isconfigured with three internal openings (FIG. 11 and construction lines200 of FIG. 5) which surround, but do not interact with, the reservoirs102-106.

[0066] Upon assembly of the biosensor, in the reservoirs 102-106, thefirst planar surface 172 of the waveguide 164 constitutes a floor orceiling (FIG. 8) of the particular reservoir, while the flow cell top100 is formed to constitute the ceiling or floor and the walls. Theorientation depicted in FIG. 8, wherein the planar surface 172 serves asa ceiling and lays level with the earth has been found to be especiallyuseful, enhancing the ability of the device to detect the presence oftarget molecules in even whole blood over a shorter period of time(e.g., five to ten minutes), especially with oscillation. However, itis, of course, understood that the flow cell assembly 190 may beoriented in any position (e.g., vertical or any angle). Angling the flowcell assembly 190 assists in removing bubbles or heavy materials awayfrom the waveguide 164, if desired. Alternatively, a dye can beincorporated into the sample solution for absorbing interfering signals.Although the reservoirs 102-106 are here shown to be generallyrectangular in shape, other shapes could be used.

[0067] The gasket 162 is preferably made of a semi-rigid material havingan index of refraction less than that of the waveguide material in thewavelength range of the exciting light. For best results, it is believedthat the index of refraction of the gasket material should be as low aspossible compared to that of the waveguide. For a waveguide made ofquartz or glass, the index of refraction would typically be from about1.46 to 1.52, higher for high-lead glass. A transparent (non-pigmented)silicon rubber (siloxane polymer) with an index of refraction of1.35-1.43 is a presently preferred material for gasket 162. TEFLON™ orTEFLON-type materials such as PTFE (polytetrafluoroethylene) or FEP(fluorinated ethylene propylene) have indices of refraction of around1.34-1.35, and may also be suitable for use as layer 196.

[0068] The other portion 198 of the gasket may be formed of an opaque(e.g., red or black) neoprene or silicon rubber material which ispreferably biologically inert although due to the metal walls, it neednot be.

[0069] D. The Flow Cell Assembly

[0070] As depicted in FIG. 18, a preferred flow cell assembly 190according to the invention generally includes a flow cell portion(generally 200, FIG. 10), gasket 162, and waveguide 164. As shown inFIG. 10, the flow cell portion includes the flow cell top 100, flow cellbottom 186, and a flow cell stage (or “flow cell platform”) 202. Asshown in FIG. 8, and as more thoroughly described herein, these threecomponents of the biosensor 190 are integrated with one another in sucha manner that excitation light enters the front lens 166 of thewaveguide 164, travels through the front lens ramp 192 and planarportion 170, and passes out of the rear lens ramp 194 and rear lens 168to the outcoupling 188.

[0071] The flow cell assembly can also include means for associating theflow cell top 100 with the flow cell bottom 186 thus sandwiching thegasket 162 and waveguide 162 therebetween. The depicted means for doingso are threaded clamping bolts 204, 206 which interact withcorrespondingly threaded holes 208, 210 in the flow cell bottom 186. Ofcourse however, equivalent means such as screws, nuts and bolts, clamps,snap fits, and the like could alternatively be used.

[0072] A waveguide registration plate 212 is shown associated with theflow cell bottom 186 (FIG. 4). The waveguide is reproducibly positionedbetween the flow cell top and bottom when aligned with the registrationplate. Also depicted is a 3-channel beam mask 214 having three aperturesfor receiving a light beam.

[0073] E. The Apparatus

[0074] Once the flow cell 200, gasket 162 and particular waveguide 164have been associated with one another, the thus formed biosensor 190 maybe used in an apparatus for performing immunoassays such asfluoroimmunoassays. As depicted in FIG. 8, such an apparatus includes alight source 216 which provides a light beam 218 which is directed bymeans of mirrors 220, 222, 224 to an optical biosensor 190. The lightsource 216 may be an argon laser or laser diode capable of emitting atcenter wavelengths of between 488 and 514.5 nm and 600 to about 900 nm(e.g., 633 nm), respectively.

[0075] The embodiment of FIG. 8 further includes a 45° angle mirror 226which is positioned for assisting in focusing the beam 218 onto theinput lens 166 of a particular biosensor 190 if desired. The biosensor190 has an optical substrate with one end 166 positioned to receivelight from beam 218. In the case of a non-integrated quartz waveguide, afocusing lens 228 is preferably positioned between angle mirror 226 andthe biosensor 190 for focusing light from beam 218 onto the end of thebiosensor. Focusing lens 228 is removable, and is depicted mounted on anX-Y translation unit so that its position may be adjusted for bestfocusing. Furthermore, the translation unit can be moved to adjust theangle Θ for waveguides of differing composition. A significant portion(in the case of the quartz waveguide, the entire portion) of the opticalsubstrate 164 is of a generally planar shape having two planar surfacesspaced by a width 176 as shown in FIG. 9, which is more thoroughlydescribed herein.

[0076] Light detection means, generally 230, are positioned to detectfluorescent light emitted from the biosensor 190. As more thoroughlydescribed herein with regard to FIG. 9, the emitted light is reflectiveof the concentration of a selected analyte in a sample. The lightdetection means 230 depicted in FIG. 8 includes a collection lens 232positioned to collect the emitted fluorescence from a directionsubstantially orthogonal to the direction of propagation of light 218through optical substrate 164.

[0077] The distance 234 between collection lens 232 and opticalsubstrate 164 is selected as known to those skilled to maximize thecollection of light emitted from the region of evanescent lightpenetration while at the same time imaging this light onto the face ofthe photodetector. The light collected by collection lens 232 is thensent to detection means 230, which responds by outputting signalsreflective of the level of collected fluorescent light.

[0078] Detection means 230 may be any type of photodetector useful todetect light in the wavelength region spanning the wavelength range ofthe emitted fluorescence, as known in the art. However, in a preferredembodiment for simultaneous multi-analyte assays, detection means 230 isan imaging-type detector providing direct imaging of each of thefluorescent signal(s) originating in the evanescent zone 236 (FIG. 9).In the apparatus of FIG. 8, detection means 230 is a CCD detector whichproduces a signal. Such imaging signal collection provides simultaneousmeasurement of multiple samples in a much simpler way than a system inwhich a separate optical element is needed to read each well or patch.The present imaging detection system also provides for collection ofemitted fluorescence directly from the evanescent zone 236, rather thanvia evanescent penetration of the fluorescence into the waveguide (FIG.9).

[0079] Alternatively, detection means 230 maybe a photomultiplier, asemiconductor photodiode, or an array of such detectors. In embodimentsother than a CCD, an array is generally preferable to a single detectorfor some purposes. With an array of small detectors, the user candetermine that the maximum fluorescence is being detected and is notinadvertently missed due to misalignment of the collection and detectionoptics. Optionally, a grating spectrograph is coupled to the CCD orother detection means to provide spectral analysis of the detectedlight. In that case, means are also provided to integrate the signalfunction around each peak to determine the total collected fluorescencefrom a sample. Alternatively, in an embodiment for use in a setting suchas in a testing laboratory, and for which all the parameters of theassay have been standardized, the spectrograph may be replaced by afilter which passes only wavelengths in the region of tracerfluorescence.

[0080] As is better seen in FIG. 9, optical substrate 164 is embodied asa planar portion of a waveguide having at least one planar surface 172spaced from a second surface 174 by a width 176. At least one surface174 is disposed in contact with a sample solution 238. Capture molecules240 are immobilized on the exposed surface 172 of the waveguide. In oneembodiment, the sample solution 238 contains a plurality of analytemolecules 242 of a selected analyte which also includes tracer molecules244. The tracer molecules can be incorporated into the sample solutionby, for example, admixing them with the sample solution beforeincorporation into the assay or by “drying” the molecules onto thewaveguide surface without actually chemically binding them to thesurface 172 (or at least not binding them permanently as would be thecase when the tracer molecules are associated with the surface by use ofa water soluble component (e.g. a soluble sugar that does not interferewith the particular interaction between capture and tracer molecules)).The capture molecules are chosen or constructed to bind to a bindingmoiety present on each of the analyte molecules 242. The tracer molecule244 is a molecule chosen to be complementary (in a binding sense) withthe capture molecules and is constructed to emit fluorescent light inresponse to stimulation by light of the appropriate wavelength (e.g., bytagging the capture molecule with a fluorescent label). The level offluorescence emitted by the tracer molecules 244 is a measure of theamount of analyte bound to the capture molecule and is therebyreflective of the concentration of analyte molecules 242 in thesolution.

[0081] When light is being propagated in the waveguide 164 andinternally reflected at the surfaces 172, 174 an evanescent light fieldis produced having an intensity curve 230 which drops off with distancefrom the surface 172, as diagramed relative to a distance axis 232 and ahorizontal axis 234 (not to scale). Evanescent light intensity variesalong axis 232, co-linear with distance. An excitation zone 236 is theonly region of the solution in which the evanescent light intensity issufficient to excite a significant or detectable fraction of tracermolecules 244 (not to scale). Tracer molecules 244 outside zone 236 willcontribute little or no induced fluorescence. Excitation zone 236 istypically between about 1000 and 2000 Å in depth.

[0082] Capture molecules 240 are reactive with the analyte molecules242, and may be whole antibodies, antibody fragments such as Fab′fragments, peptides, epitopes, membrane receptors, whole antigenicmolecules (haptens) or antigenic fragments, oligopeptides,oligonucleotides, mimitopes, nucleic acids and/or mixtures thereof.Capture molecules 240 may also be a receptor molecule of the kindusually found on a cell or organelle membrane and which has specificityfor a desired analyte, or a portion thereof carrying theanalyte-specific-binding property of the receptor.

[0083] The capture molecules 240 may be immobilized on the surface 172by any method known in the art. However, in the preferred embodiment,the capture molecules are immobilized in a site-specific manner. As usedin this application, the term “site-specific” means that specific siteson the capture molecules are involved in the coupling to the waveguide,rather than random sites as with typical prior art methods. Int'l Publ.No. 94/27137, which has been previously referenced, details methods forsite-specific immobilization of capture molecules to the surface of theoptical substrate by means of a protein-resistant coating on thesubstrate.

[0084] The waveguide can be designed so that multiple (e.g., four)different assays can be performed on the same sample. This isaccomplished by immobilizing different types of capture antibodies ondifferent regions of the waveguide, a process referred to as patterning.Three different patterning methods appear suitable for immobilizingantibodies to the polystyrene sensors—gasketed multi-well coating tray,liquid jet printing and photolithography. In the former, a machinesimilar to an ink jet printer is used to spray reagents onto a specificregion of the waveguide; in the latter, ultraviolet light is used tophotochemically cross-link antibodies to selected regions.

[0085] One immobilization chemistry is based on physical adsorption ofantibodies to the waveguide. In one method, an antibody is brieflyexposed to acidic conditions just prior to immobilization. It has beenshown that this acid pre-treatment step improves the antigen-bindingcapacity (AgBC) of immobilized antibodies by up to 3-fold in some cases.This immobilization chemistry is relatively simple and compatible withgasketed multi-well coating tray or liquid jet printing technology, butin some cases it exhibits a higher degree of non-specific binding thanother methods.

[0086] The other two immobilization chemistries are based on a family oftri-block polymers of the form PEO-PPO-PEO, where PEO stands forpoly(ethylene oxide) and PPO stands for poly(propylene oxide). Thesesurfactants are sold under the trade name PLURONICS and come in avariety of chain lengths for both the PEO and PPO blocks. The PPO blockis significantly more hydrophobic than the PEO blocks and adsorbsreadily to non-polar surfaces such as polystyrene, leaving the PEOblocks exposed to bulk solution. The free ends of the PEO chains exhibithigh mobility, literally sweeping proteins away from the surface.

[0087] In both the second and third immobilization chemistries, thesurface of the waveguide is coated with pluronics before attachment ofantibodies, but the two chemistries differ in how the antibodies areattached. In the second chemistry a photochemical cross-linking agent isused to conjugate antigen-binding fragments (Fab′) to the PEO blocks,making this method suitable for patterning by photolithography. In thethird chemistry, Fab′ fragments are attached to pluronics using achemical cross-linking agent, making this method compatible withgasketed multi-well coating tray or liquid jet patterning. Thephotochemical cross-linking method was evaluated with two differentPLURONICS (F108 & P105) and two different photochemical crosslinkers(BPM and BPIA). While acceptable levels of total antigen binding can beobtained with all four pairwise combinations, an unacceptable level ofNSB may be obtained when antibodies are immobilized to F108 using theBPIA crosslinker. The other three pairwise combinations give very lowlevels of NSB (about 1.5% of total binding). Furthermore, the P105/BPMpair is especially good, giving an undetectable level of NSB.

[0088] In FIG. 9, a competition assay scheme is depicted (also termed adisplacement assay). However, as will be apparent to the skilled person,alternate assay schemes such as sandwich assays may be performed withthe present apparatus. See, e.g. U.S. Pat. Nos. 4,376,110 and 4,486,530to Hybritech, Inc.

[0089] In the embodiment of FIG. 9, the competition immunoassay hastracer molecules 244 constructed such that the capture molecules 240will bind tracer molecules 244 in place of analyte molecules 242. Higherconcentrations of analyte molecules 242 will cause most of the tracermolecules 244 to be displaced into the surrounding solution from capturemolecules 240, thus reducing the number of tracer molecules withinexcitation range 236 of the substrate 164. This reduced binding oftracer molecules in turn reduces the amount of fluorescence. Incontrast, lower concentrations of analyte molecules 242 will allowtracer molecules 244 to bind to capture molecules 240, and thus to beheld within the excitation range 236.

[0090] In tests conducted with the point-of-care cardiovascular markerCK-MB (associated with acute myocardial infarction) on both plasma andwhole blood, the results were comparable (taking into considerationdiffusion and viscosity differences).

[0091] In the embodiment of the apparatus of FIG. 8, measurements offluorescence are made by spectroscopy. Fluorescence detection was donewith a monochromator (SPEX Industries, Inc., Model 1680C) and a CCD(Photometrics Ltd. Series 200, or CH-250). Alternatively, light source216 can be any light source emitting at the wavelength desired forexcitation of selected fluorescent dyes. Also, once an assay procedurehas been validated and standardized, it may not be necessary to measurethe fluorescence spectrum or spatial distribution of fluorescence. Thedetection means may be simplified in accordance with the minimumrequirements of the assay.

[0092] In another alternate embodiment, light source 216 is a laserdiode emitting in the red wavelength region of 600-700 nm which iscommercially available. The laser diode may provide about 12 milliwattsof power with a peak emission wavelength of about 635 nm. Laser diodesemitting at 633 nm are also available and can be used. For an embodimentusing a wavelength in this region, it is necessary to use dyes such ascyanine dyes, whose fluorescence can be stimulated by excitation withwavelengths in the red spectral region. An example of such a dye is thefluorescent dye CY5, available from Biological Detection Systems, Inc.,Pittsburgh Pa. (catalog no. A25000). The CY5 dye can be conjugated tothe desired tracer molecule by the manufacturer's instructions and/orwith a kit available from BDS. A second dye, CY7, may also be suitable.The dyes and methods for conjugating are also characterized in the paperby Southwick, P. L., et al., titled “Cyanine Dye LabellingReagents—Carboxymethylindocyanine Succinimidyl Esters”, Cytometry11:418-430 (1990). The use of laser diodes as a light source permits thebiosensor and waveguide to be formed of plastic, which considerablyreduces the expense of manufacture and facilitates the integral moldingof the semi-cylindrical lens with the waveguide and reservoirs.

[0093] Different labels can be used which emit light at differentwavelengths if desired. In such a circumstance, different types ofcapture molecules (e.g, antibodies reactive with different antigens) canbe immobilized to the surface so that the waveguide can be used todetect more than one molecule to be detected. In such a case, multiplewavelengths can be detected by multiplexing the signal from thewaveguide.

[0094]FIG. 24 stylistically illustrates simultaneous wavelength- andspatially-resolved detection of fluorescence emitted from a waveguidesensor using different capture molecules (Capture Ab₁, Capture Ab₂,Capture Ab₃, . . . Capture Ab_(x)), tracer molecules (Tracer Ab₁, TracerAb₂, Tracer Ab₃, . . . Tracer Ab_(x)), and labels (F₁, F₂, F₃, . . .F_(x)) with the purpose of detecting different analytes of interest(Analyte₁, Analyte₂, Analyte₃, . . . Analyte_(x)) in a sample solution238.

[0095] In the depicted embodiment, the device works as otherwise hereindescribed, but each tracer molecule (e.g, Tracer Ab₁, Tracer Ab₂, TracerAb₃, . . . Tracer Ab_(x)) is labeled with a different coloredflourophore (F₁, F₂, F₃, . . . F_(x)).

[0096] The waveguide is illuminated by one or more different wavelengthsof light 218 appropriate to excite all the fluorophores located withinthe evanescent region of the waveguide. In one configuration, theemissions from the different fluorophores are distinguished usingbandpass filters. Light rays 248, 249 and 250 are emitted from therespective labels on the tracer molecules. This light then passesthrough a lens 252 collimates the emitted light onto a band pass filter254 selective for the wavelength emitted by the particular tracermolecule label, in the depicted case, Tracer Ab₁. A filter switchingmember, such as a wheel 256, houses, for example, three different bandpass filters—each selective for a different fluorophore label. Thus,only the light rays 248 emitted by Tracer Ab₁ pass through the filter254. If spatial resolution is desired in addition to wavelengthselection, the light 248 passing through the filter 254 passes through asecond lens 258 which images the light 248 onto a spatially-resolvedphotodetector 260 such as a CCD or diode array. If only wavelengthresolution is desired, the photodetector 260 may be a singlespatially-integrating device, and lens 258 may be optionally omitted

[0097] Alternatively, the wavelength selectivity may be accomplished byone of several means instead of a filter wheel, such as employing adiffraction grating, a prism, or an acousto-optical modulator toangularly separate the different emitted wavelengths and thus directthem to separate individual photodetector elements whose outputs arerepresentative of the signal strengths in each wavelength band. Inanother arrangement which avoids the use of the rotating filter wheel,stationary beam splitters are employed to direct portions of the emittedlight through stationary filters placed in front of individualphotodetector elements.

[0098] Alternatively, if the excitation wavelengths of the differentfluorophores are sufficiently separated without appreciable overlap, thelight source may sequence in time through each excitation wavelength.The emitted light at any given time is related to the signal strength ofthe fluorophore set whose excitation wavelength is chosen at thatparticular time, and no further wavelength selective devices, such asfilters, are needed.

[0099] The invention is further explained by the following illustrativeexamples.

EXAMPLE I

[0100] A waveguide with integrated lenses, such as that depicted inFIGS. 6 & 7, was injection molded in a clean environment from atransparent, general purpose polystyrene. The waveguide had a length of38 mm, and a width of 25 mm. The thickness 176 of the planar surface 170was a consistent 0.5 mm. The ridge or “shelf” had a height of 1.3 mm.The front lens and rear lens had bottom edges co-planar with theirrespective centers of curvature. The front lens horizontal angle 262 wasabout 15°. The rear lens horizontal angle 264 was about 19°. The radiiof curvature of the front and rear lenses were about 3.2 mm and 1.6 mmrespectively. The mean angle Θ of the front lens was 21°. The mean angleof the rear lens was about 24°.

EXAMPLE II

[0101] A flow cell top, such as that depicted in FIGS. 1-3, and 8, wasmade of hard black anodized 6061-T6 aluminum. It contained threereservoirs each of which had a 0.25 mm (0.010 in.) thick wallsurrounding it, a flat floor in middle, two half-capsule shaped recessesat either end 1.6 mm ({fraction (1/16)} in.) in width, and ports 1.6 mm({fraction (1/16)} in.) in diameter running into the center of eachrecess. The ports opened into a #10-32 (standard thread, not NPT)connector which ran out to the opposite face of the flow cell and was5.1 mm (0.200 in.) deep. A 90° countersink 266 (FIG. 2) was given at thesurface of the port connector (a plastic barbed tubing connector screwedinto the port connector and sealed on the countersink). On both sides ofthe array of reservoirs were two raised platforms which were referred toas lands 268, 270. Each land had three holes running through thethickness of the part. The four #31 clamping holes 272, 274, 276, 278were formed (i.e., drilled through). The two apertures 178, 180 weredrilled and reamed to achieve a close sliding fit with 2.4 mm ({fraction(3/32)} in.) nominal four-sided pins 182, 184 press fit into the secondframe member 186.

EXAMPLE III

[0102] A gasket 162, such as that depicted in FIGS. 4, 5, and 11, wasmade as a composite structure laminated from 1.6 mm ({fraction (1/16)}in.) silicone rubber sheeting and 0.076 mm (0.003 in.) self-adhesive FEPfilm (total thickness: 1.676 mm (0.066 in.) nominal). Its outerdimensions were about 25 mm (1 in.) by 25.40 mm (1.000 in.) and it hadthree internal openings which corresponded to the flow cell reservoirs.The gasket was produced using a waterjet cutter and was seated on theflow cell such that the FEP layer faced away from the flow cell surface.

EXAMPLE IV

[0103] A second member 186, such as that depicted in FIGS. 4, 10, 12, 13and 18 was made from hard black anodized 6061-T6 aluminum. It containedthree internal openings 280, 282, 284 which corresponded to the threereservoirs 102, 104, 106 of the flow cell 100 (but were slightlylonger). The internal openings were positioned in a shallow depression(0.46 mm (0.018 in.) deep) 286 which seated the waveguide, and allowevanescent light emitted from any reacting tracer molecules on thewaveguide surface to be detected by the detection means 230. As with theflow cell 102, two lands 288, 290 resided on either side of the internalopenings, each land having three holes. Four clamping holes (e.g., 208,210) were drilled through and tapped to #4-40 to receive thumb screws.Two apertures were drilled through to receive a 3.2 mm (⅛ in.) nominaldowel which was press fit into the hole. The dowel was stainless steeland projected approximately 7 mm (0.280 in.) above the top surface and6.6 mm (0.260 in.) below the bottom surface of the second member. Theexposed dowel was machined down to 2.4 mm ({fraction (3/32)} in.)nominal diameter and was squared off to produce a low-friction locatingpin 182, 184. The bottom aspect of the secondary member was milled outto provide a single large window 292 for emitted fluorescence from thewaveguide. The front surface of the secondary member contains twomounting holes 294, 296 #2-56 drilled and tapped to a depth of about 4mm (0.15 in.) to fasten the flow cell registration plate 212. Theregistration plate, depicted in FIGS. 14, is a simple U-shaped bracketwhich was produced from 1.6 mm ({fraction (1/16)} in.) nominal 6061-T6aluminum plate. It contained two#42 holes which corresponded to theholes on the front of the second member. The purpose of the registrationplate was to provide a lateral hard-stop for the waveguide duringclamping into the flow cell. The upright arms of the part contact thewaveguide at the outer edges of the input coupling lens while allowingunimpeded coupling with the incoming laser beam.

EXAMPLE V

[0104] A stage 202, best depicted in FIGS. 15-17, is a plate-likestructure which was made from hard black anodized 6061-T6 aluminum. Thereceiving site 298 of the part was down-stepped and contains a singlerectangular internal opening. Three #2-56 drilled and tapped holes 300,302, 304 were positioned on the front of the part which were used tofasten a laser beam mask (not shown). Two #10-32 holes 306, 308 weredrilled to a depth of 19 mm (0.750 in.) on the right side of the part tomount the stage to the test apparatus. The internal opening had beveledfront 310 and rear 312 sides. Located on either side of the window weretwo 2.4 mm ({fraction (3/32)} in.) apertures (analogous to those in theflow cell) which allowed the clamped flow cell and second member to bemounted to the stage 202.

EXAMPLE VI

[0105] The waveguide and integrated lenses of EXAMPLE I, the flow celltop of EXAMPLE II, the gasket of EXAMPLE III, the second member andregistration plate of EXAMPLE IV, and the stage of EXAMPLE V wereassociated as in FIG. 4. A hard, black anodized coating was added to theparts with a nominal build-up of 0.0025 mm (0.001 in.), however, theassembly 190 was checked as much as possible prior to anodization tomaximize the probability of proper fit.

[0106] The gasket was cut to correspond to the outside dimensions of thethree reservoirs 102-106 of the flow cell top 100. The silicone rubbersurface contacted the flow cell and the FEP surface contacted thewaveguide when the assembly was clamped. Any flash present on the gasketwhich interfered with seating or which came over the top of the wallswas carefully trimmed back with a razor knife (the top of the dam wasexposed to the surface of the waveguide, but did not touch it; flashfrom the gasket can interfere with proper clamping).

[0107] The waveguide 164 was seated in the shallow depression in thesecond member 186. The waveguide fit into the depression with minimallateral movement, but without compression or pinching. A small amount(e.g., less than about 0.1 mm (0.003 in.)) of lateral movement wasacceptable. If pinching occurred, additional milling to the walls of thedepression was necessary to allow proper seating.

[0108] To insure that the waveguide 164 was reproducibly positioneddirectly beneath the flow cell, it was butted up against theregistration plate 214 after being seated in the secondary member 186.Contact with the registration plate 214 was only at the outermostcorners of the front lens; no contact with the injection mold stub onthe underside of the front lens occurred (injection mold may be designedto place resultant stub at an alternate location). The front of thesecond member may need to be milled to ensure the waveguide sitsdirectly beneath the flow cell when in contact with the registrationplate.

[0109] After seating the gasket into the flow cell and positioning thewaveguide on the second member, the flow cell was mated with the secondmember by engaging the locating pins into the apertures in the flowcell. When fully engaged, but without adding additional clamping force(i.e., the gasket was not compressed), there was a 0.15 mm (0.006 in.)gap between the lands of the flow cell and the lands of the secondmember. When fully clamped with four thumb screws such that the landsare in contact, the gasket is compressed 0.15 mm (0.006 in.). The flowcell and second member readily separated using manual force; no stickingoccurred, but a thin coat of lubricant may be used on the pins ifnecessary. It may be desirable to slightly countersink the press fithole on the second member and/or the aperture on the flow cell to avoidburrs or bulges which might impair mating of the two parts.

[0110] The locating pins from the bottom of the second member readilyaligned and fit into the apertures on the stage. No perceptible playexisted between the parts when mated. As with the flow cell and secondmember fit, the second member and stage readily separated using moderatemanual force.

EXAMPLE VII

[0111] As shown in FIG. 20, onto a polystyrene sheet waveguide 316(Scenic Materials, 125 μm thick, n=1.60) was adhered a piece ofholographic diffraction grating 314 (Edmund Scientific #43226, 2400g/mm, Al/N₉F₂ coated) with an index-matching cement 320 consisting of10% polystyrene chips dissolved in toluene. Incoupling into theresulting waveguide was achieved with a helium-neon laser at anapproximately 5° angle. The resulting coupling structure was thinnerthan a molded lens input coupler.

EXAMPLE VIII

[0112] A. Waveguide Fabrication

[0113] Thin film channel waveguide layers of SiON were formed on gratingetched quartz wafers in a manner such as that described in Walker et al.“Corning 7059, silicon oxynitride, and silicon dioxide thin-filmintegrated optical waveguides: In search of low-low, non-fluorescentreusable glass waveguides”, Appl. Spectrosc., 46: 1437-1441 (1992).Briefly, the wafers were introduced into a plasma impulse chemical vapordeposition (“PECVD”) chamber (Texas Instruments) operating at 300° C.,50 W, and 1.25 Torr. The gases used were SiH₄, silane, nitrogen,ammonia, and nitrous oxide. SiON films were produced at a depositionrate of approximately 590 Å/minute for 25.42 min., yielding a filmthickness of 1.5μ. As described in Plowman et al., “Femtomolarsensitivity using a channel-etched thin film waveguidefluoroimmunosensor”, Biosensors & Bioelectronics, 11: 149-160 (1996),nine parallel 1 mm×65 mm channels are formed when etched into the SiONfrom an additional layer of photoresist. The resulting waveguide waferswere diced (Disco DAD-2H/6) into three rectangular pieces measuringabout 2.5 cm×7.8 cm each with three waveguiding channels (although inthis EXAMPLE, grating waveguides without channels were employed).

[0114] B. Grating Fabrication

[0115] Quartz wafer substrates (Hoya, Woodcliff Lake, N.J., QZ4W55-325-UP) measuring 100 mm in flat length and 0.5 mm in thicknesswere cleaned at room temp. in a 6% solution of H₂O₂ in H₂SO₄. The wafersthen received a HMDS vacuum vapor prime, were spin-coated withphotoresist (Shipley SNR 200, MA, USA) at a rate of 4200 rpm for 1 min.to produce a film approximately 0.7 mm thick and then soft-baked at 100°C. for 1 min. The negative resist was exposed to define 0.7μ periodicgroove patterns using ultraviolet light (248 nm KrF excimer laser, LaserStepper GCA-ALS, Tukesbury, Mass., USA) with a chrome-quartz mask(DuPont, Kokomo, Ind., USA) at a dose of 20 mJ/cm². The wafers receiveda post-exposure bake at 130° C. for 90 sec. Photoresist was developed ina solution of Shipley MF 312 (MA, USA) at a normalization of 0.17, thenspun dry. A 60 sec. 100° C. post-development bake was then performed.The resulting photoresist pattern was reactive ion-etched (8110 ReactiveIon Etcher, AME, Santa Clara, Calif., USA) with O₂ and CHF₃ gases at anetch rate of 450 Å/min. Etch times of 13.33, 17.77 and 22.22 min. wereemployed to produce grating etch depths of 0.6, 0.8, and 1.0μ givingaspect ratios of 0.8, 1.0, and 1.4, respectively. Residual photoresistwas removed by O₂ plasma.

[0116] C. Testing

[0117] When used as light couplers for thin film IOWs, the gratings wereroughly half as efficient as prisms in coupling laser light (the lightsource being a 2 mW He-Ne laser (632.8 nm, 10 mW maximum, Melles-Groit,Uniphase, Manteca, Calif., USA). Both approaches (i.e. prism andgrating) detected samples with femtomolar concentration abovebackground.

EXAMPLE IX

[0118] An evanescent wave assay apparatus of the instant invention wascompared with a standard ABBOTT STAT CK-MB IMx system.

[0119] A. Clinical Samples

[0120] 63 clinical samples (submitted by physicians for hospital CK-MBtesting) were obtained, and were assayed for CK-MB using the ABBOTT IMxsystem. The values thus obtained for each clinical sample was noted.Each sample was aliquoted into 0.550 ml sample size, assigned a lotnumber, and stored at −20° C.

[0121] B. Instrumentation/Assay standardization

[0122] The samples were then assayed on a CK-MB system utilizing thebiosensor 190 of the instant invention. CK-MB specificity was determinedby spiking CK-MB stripped plasma (solid-phase absorption) with 1000ng/ml CK-MM and CK-BB. Cross-reactivity with CK-MM was less than 0.1%.Standards were prepared by addition of a known mass quantity ofrecombinant CK-MB (Genzyme).

[0123] C. Monoclonal Antibodies

[0124] Monoclonal antibodies were as described by J. Ladenson, ClinicalChemistry, vol. 32, pp. 657-63 (1986). The capture antibody was coatedonto the waveguide's surface with 2 hour incubation at room temp., at aconcentration of 0.1 micromolar (diluted in PBSA buffer). After theincubation, each waveguide was washed once with PBSA, and then incubatedwith 1 ml of postcoating solution (0.5% bovine serum albumin(“BSA”)/0.1% trehalose/PBSA) at room temp. for 1 hour. The post-coatingsolution was discarded and the waveguides dried in a vacuumed desiccatorfor an hour.

[0125] D. Specific Performance

[0126] The correlation of an apparatus according to the instantinvention with the ABBOTT IMx assay for an CK-MB assay is graphicallydepicted in FIGS. 21 & 22. The results showed the two sets of values tobe comparable at a correlation coefficient of 0.98, and the samplepopulation contained a higher proportion of samples below 50 ng/ml, therange in which the critical decision point for clinical evaluation hasbeen established in the clinical laboratory environment. FIG. 21 plotsthe value obtained with the instant invention (y axis, in ng CK-MB/ml)against the ABBOTT IMx value on the x-axis (also ng CK-MB/ml). FIG. 22depicts the double logarithm plot of the data from FIG. 21 (again,instant invention y-axis, ABBOTT IMx x-axis).

EXAMPLE X

[0127] The preceding example was continued to further establish theutility of the system, and to incorporate more extensive studies ofknown interfering substances (e.g., hemoglobin and bilirubin), knownCK-MB concentrations were analyzed (20 ng/ml CK-MB into human plasma),with concentrations of hemoglobin (15 mg/ml) and bilirubin (1 mg/ml)known to interfere with immunofluourescence assays. The system was stillable to detect 100% of the CK-MB.

[0128] Characteristics of the described and illustrated embodiments areintended for illustrative purposes, and are not to be consideredlimiting or restrictive. It is to be understood that various adaptationsand modifications may be made by those skilled in the art to theembodiments illustrated herein, without departing from the spirit andscope of the invention, as defined by the following claims thereof.

What is claimed is:
 1. An apparatus for analyzing a biological fluid,comprising: a waveguide, comprising: a planar portion with oppositefirst and second planar surfaces and opposite front and rear ends, saidplanar portion capable of transmitting light from said front end to saidrear end; at least one ramp comprising a lens for receiving light, saidat least one ramp in optical communication with and extending from thefront end of said planar portion, a surface of said at least one rampbeing oriented at an angle other than 0° from a plane of said planarportion; at least one type of capture molecule immobilized relative tosaid first planar surface of said planar portion of said waveguide; alight source oriented so as to direct at least one wavelength of lightinto said first lens; and a light detector oriented to receive lightemitted through said second planar surface.
 2. The apparatus of claim 1,wherein said first planar surface is configured to receive a sample orsolution comprising a sample.
 3. The apparatus of claim 1, wherein thebiological fluid comprises at least one of blood cells and portions ofblood cells.
 4. The apparatus of claim 1, wherein said first planarsurface of said planar portion of said waveguide is oriented to face inan at least partially downward direction.
 5. The apparatus of claim 4,wherein said planar portion is oriented substantially horizontal.
 6. Theapparatus of claim 4, wherein said planar portion is orientednonhorizontally.
 7. The apparatus of claim 4, wherein said first planarsurface is configured to receive a sample or a sample solutioncomprising at least one of blood cells and portions of blood cells. 8.The apparatus of claim 1, wherein said planar portion is orientedsubstantially vertically.
 9. The apparatus of claim 1, wherein saidfirst planar surface of said planar portion of said waveguide isoriented to face in an at least partially upward direction.
 10. Theapparatus of claim 9, wherein said planar portion is orientedsubstantially horizontal.
 11. The apparatus of claim 9, wherein saidplanar portion is oriented nonhorizontally.
 12. The apparatus of claim1, wherein said waveguide comprises an optical plastic.
 13. Theapparatus of claim 1, wherein said angle comprises an angle of fromabout 15° to about 32°.
 14. The apparatus of claim 1, wherein at leastone type of capture molecule is arranged over said first planar surfaceof said planar portion of said waveguide in an array of reaction sites.15. The apparatus of claim 1, comprising capture molecules that reactselectively with different, corresponding selected analytes.
 16. Theapparatus of claim 15, wherein said capture molecules are arranged oversaid first planar surface of said planar portion of said waveguide indiscrete reaction sites from one another.
 17. The apparatus of claim 16,wherein said discrete reaction sites are arranged over said first planarsurface in an array.
 18. The apparatus of claim 1, further comprising: aflow cell cover configured to be positioned adjacent to said firstplanar surface of said planar portion of said waveguide, said flow cellcover forming at least one wall of at least one reservoir adjacent tosaid first planar surface.
 19. The apparatus of claim 18, furthercomprising: an inlet in fluid communication with said at least onereservoir to facilitate introduction of a sample over at least a portionof said first planar surface.
 20. The apparatus of claim 1, wherein saidwaveguide comprises a single material layer.
 21. The apparatus of claim20, wherein said single material layer comprises an optical plastic. 22.The apparatus of claim 1, further comprising an optical substrate thatsupports said waveguide.
 23. The apparatus of claim 22, wherein saidoptical substrate comprises at least one of polystyrene,polymethylmethacrylate, polyvinyl chloride, polyimide, polyester,polyurethane, an organically modified ceramic, a polymer of diethyleneglycol bisallyl carbonate, allyldiglycolcarbonate, and polycarbonate.24. The apparatus of claim 22, wherein said waveguide comprises at leastone of an optical plastic, TiO₂, SiO₂, ZnO₂, Nb₂O₂, a silicon nitride, asilicon oxynitride, Ta₂O₅, HfO₂, and ZrO₂.
 25. A biosensor, comprising:a waveguide, comprising: a planar portion with opposite first and secondplanar surfaces, said planar portion capable of transmitting light fromsaid front end to said rear end; and at least one type of capturemolecule immobilized relative to said first planar surface of saidplanar portion of said waveguide, said at least one type of capturemolecule arranged over said first planar surface in a plurality ofdiscrete reaction sites.
 26. The biosensor of claim 25, wherein saidplurality of discrete reaction sites comprises an array of reactionsites.
 27. The biosensor of claim 25, comprising capture molecules thatreact selectively with different, corresponding selected analytes. 28.The biosensor of claim 25, wherein said capture molecules that reactselectively with different, corresponding selected analytes are arrangedin different reaction sites of said plurality of discrete reaction sitesfrom one another.
 29. The biosensor of claim 25, further comprising: aflow cell cover positioned adjacent to at least a portion of said firstplanar surface of said planar portion, said flow cell cover forming atleast one wall of at least one reservoir adjacent to said first planarsurface.
 30. The biosensor of claim 29, further comprising: an inlet influid communication with said at least one reservoir to facilitateintroduction of a sample over at least a portion of said first planarsurface.
 31. The biosensor of claim 25, further comprising: at least oneramp comprising a lens for receiving light, said at least one ramp inoptical communication with and including a surface that extends from thefront end of said planar portion at an angle other than 0° from a planeof said planar portion.
 32. The apparatus of claim 25, wherein saidwaveguide comprises a single material layer.
 33. The apparatus of claim32, wherein said single material layer comprises an optical plastic. 34.The apparatus of claim 25, further comprising an optical substrate thatsupports said waveguide.
 35. The apparatus of claim 34, wherein saidoptical substrate comprises at least one of polystyrene,polymethylmethacrylate, polyvinyl chloride, polyimide, polyester,polyurethane, an organically modified ceramic, a polymer of diethyleneglycol bisallyl carbonate, allyldiglycolcarbonate, and polycarbonate.36. The apparatus of claim 34, wherein said waveguide comprises at leastone of an optical plastic, TiO₂, SiO₂, ZnO₂, Nb₂O₂, a silicon nitride, asilicon oxynitride, Ta₂O₅, HfO₂, and ZrO₂.
 37. A biosensor for use in anapparatus which uses light to analyze a sample solution comprising abiological liquid, the biosensor comprising: a waveguide including atleast one planar surface; a taper extending from said at least oneplanar surface and optically associated therewith, said taper configuredto receive and convey light into said waveguide; capture moleculesassociated with said at least one planar surface; a flow cell coverpositioned adjacent to and spaced apart from said at least one planarsurface and, with said at least one planar surface, defining at leastone reservoir for containing the biological liquid; and at least aninlet in fluid communication with said at least one reservoir tofacilitate introduction of the sample solution therein so as to permitthe biological liquid to contact said capture molecules for interactiontherewith.
 38. The biosensor of claim 37, wherein said flow cell covercomprises a flow cell top.
 39. The biosensor of claim 37, furthercomprising: at least one outlet in fluid communication with said atleast one reservoir.
 40. The biosensor of claim 39, wherein said atleast one outlet is configured to be contacted by sample solution withinsaid at least one reservoir.
 41. The biosensor of claim 39, wherein saidat least one inlet, said at least one reservoir, and said at least oneoutlet are in fluid-tight communication.
 42. A method for analyzing asample solution comprising a biological liquid, the method comprising:providing a biosensor comprising a waveguide and a plurality of capturemolecules on a surface of said waveguide; exposing said capturemolecules to the sample solution, the biological fluid of which maycomprise molecules of at least one selected analyte; adding tracermolecules to said solution, each tracer molecule including a sitecapable of binding with at least a portion of a complementary capturemolecule or at least a portion of said analyte, each tracer moleculeincluding a component that emits fluorescent radiation of an emissionwavelength when exposed to radiation of an excitation wavelength;introducing light of said excitation wavelength into said waveguide; anddetecting light of said emission wavelength passing through anothersurface of said waveguide.
 43. The method according to claim 42, furthercomprising: determining an amount of said at least one selected analytebased on said detecting.
 44. The method according to claim 43, whereinsaid determining comprises determining amounts of a plurality ofselected analytes based on said detecting.
 45. The method according toclaim 42, wherein said detecting comprises positioning a light detectorwithin a cone of collection angles having an axis oriented substantiallyorthogonal to a plane of said waveguide.
 46. The method according toclaim 42, wherein said providing said waveguide comprises providing saidwaveguide with said capture molecules arranged in discrete reactionsites.
 47. The method according to claim 46, wherein said providing saidwaveguide comprises providing said waveguide with said discrete reactionsites organized in an array.
 48. The method according to claim 47,wherein said providing said waveguide comprises providing said waveguidewith capture molecules of at least one reaction site of said arrayhaving specificity for a different selected analyte than anotherselected analyte for which capture molecules of at least anotherreaction site have specificity.
 49. A method for fabricating a biosensorcomprising: forming a waveguide to have at least one substantiallyplanar surface; and immobilizing capture molecules over said at leastone substantially planar surface, said capture molecules being arrangedover said at least one substantially planar surface at a plurality ofdiscrete reaction sites.
 50. The method according to claim 49, whereinsaid immobilizing comprises immobilizing said capture molecules oversaid at least one substantially planar surface in an array of reactionsites.
 51. The method according to claim 49, wherein said immobilizingcomprises: immobilizing capture molecules having specificity for a firstselected analyte at a first reaction site of said plurality of discretereaction sites; and immobilizing capture molecules having specificityfor a different, second selected analyte at a second reaction site ofsaid plurality of discrete reaction sites.
 52. The method according toclaim 49, wherein said immobilizing comprises patterning said capturemolecules over said at least one substantially planar surface.
 53. Themethod according to claim 49, wherein said forming comprises formingsaid waveguide to comprise a single material layer.
 54. The methodaccording to claim 53, wherein said forming said waveguide comprisesforming said waveguide from an optical plastic.
 55. The method accordingto claim 49, wherein said forming comprises forming said waveguide on anoptical substrate.
 56. The method according to claim 55, comprisingforming said optical substrate from at least one of polystyrene,polymethylmethacrylate, polyvinyl chloride, polyimide, polyester,polyurethane, an organically modified ceramic, a polymer of diethyleneglycol bisallyl carbonate, allyldiglycolcarbonate, and polycarbonate.57. The method according to claim 55, comprising forming said waveguidefrom at least one of an optical plastic, TiO₂, SiO₂, ZnO₂, Nb₂O₂, asilicon nitride, a silicon oxynitride, Ta₂O₅, HfO₂, and ZrO₂.